MicroRNA-200 Family Members Differentially Regulate Morphological Plasticity and Mode of Melanoma Cell Invasion

Marshall CJ (2010) MicroRNA-200 Family Members Differentially Regulate Morphological Plasticity and Mode of Melanoma Cell Invasion. PLoS ONE 5(10): e13176. doi:10.1371/journal.pone.0013176 MicroRNA-200 Family Members Differentially Regulate Morphological Plasticity and Mode of Melanoma Cell Invasion Ilan Elson-Schwab 0 Anna Lorentzen 0 Christopher J. Marshall 0 Erik H. J. Danen, Leiden University, Netherlands 0 Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research , London , United Kingdom Background: A functional role of microRNAs (miRNAs or miRs) in neoplasia and metastasis is becoming clear, and the miR200 family has received much attention for potentially regulating tumor progression. The miRNAs of this family have been shown to suppress epithelial-mesenchymal transition, and their down-regulation in some tumors promotes invasion and metastasis. Interestingly, while miR-200 is down-regulated in some cancers, it is up-regulated in others. Principal Findings: We show that levels of miR-200 are increased in melanoma cell lines compared to normal melanocytes and that miR-200 family members play a role in determining modes of tumor cell migration. Individual tumor cells can invade in either elongated, ''mesenchymal-type'' or rounded, ''amoeboid-like'' modes and these two modes of invasion are inter-convertible [1]. In melanoma cell lines, expression of miR-200 members does not suppress invasion but rather leads to a switch between modes of invasion. MicroRNA-200c results in a higher proportion of cells adopting the rounded, amoeboid-like mode of invasion, while miR-200a results in a protrusion-associated elongated mode of invasion. Functional target identification studies suggest that the morphological effects of miR-200c may be mediated by reduced expression of MARCKS, which has been linked to formation of cell protrusions. In contrast miR-200a reduces actomyosin contractility, a feature of rounded morphology. Significance: Overall our findings call into question the general role of miR-200 in suppressing invasion and metastasis, and highlight novel distinguishing characteristics of individual miR-200 family members. - Funding: This work was supported by Cancer Research UK. C.J.M. is a Cancer Research UK Gibb Life Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Melanoma is a highly aggressive human cancer refractory to most treatments. Progression from benign hyperplastic melanocytes to more aggressive disease occurs when tumor cells begin to break down and invade through the basement membrane, and subsequently migrate into the collagen-rich dermis [2]. It has become clear that cancer cells have multiple modes of cell migration during tissue invasion: collective, individual elongated or mesenchymal-type, and individual rounded or amoeboidtype [1,3,4]. The latter two modes of individual cell migration are determined in large part by the balance of Rho and Rac small GTPase signalling. The mesenchymal mode is driven by Rac activation, and involves extensive protrusions and proteolytic activity [1]. The rounded amoeboid-type on the other hand is associated with a high degree of actomyosin contractility, membrane blebbing and squeezing through matrices. The amoeboid mode is favored by high Rho/ROCK signalling to elevate actomyosin contractility [1] and is not dependent on extracellular protease activity [4]. Importantly, there is negative feedback between these two signalling pathways, with Rho-kinase inhibiting the mesenchymal mode and Rac inhibiting the rounded form of migration [1]. This interplay allows for dynamic signalling and survival pathway dependence, and plasticity or switching between different morphologies allows cancer cells to invade using distinct pathways to adapt to different environments [4]. Cancer cell morphology can be modulated by microRNA (miRNA or miR) activity [5,6,7]. MicroRNAs are 2024 nucleotide non-coding RNAs that regulate gene expression by targeting the 39 untranslated region of target mRNA transcripts for degradation and/or translation inhibition. Target specificity is directed by sequence complementarity to the microRNA particularly in the 29-89 seed region - and families have been identified based on miRs that have highly similar or identical seed sequences [8]. Of particular interest in the field of metastasis, the miR-200 family has been shown to regulate epithelial-mesenchymal transition (EMT) and cell migration in a variety of cancer cell lines: miR-200a, -200b, -200c and -141 promote E-cadherin-based junction formation and inhibit cell migration in Boyden-type transwell chambers [9,10,11,12]. It appears that the miR-200 family is targeting Zeb transcriptional regulators [5], particularly Zeb1 [9,10,11,12,13], preventing the repression of E-cadherin expression by Zeb proteins. Because miR-200 levels are decreased in more aggressive metaplastic breast as compared with ductal tumors [10], and EMT is associated with disruption of cell-cell adhesion and the acquisition of migratory behavior, it has been suggested that the miR-200 down-regulation is involved with the progression of cancer through promoting EMT and cell invasion. However, while the expression of miR-200 family members is down-regulated in some types of cancer, these microRNAs are over-expressed in other cancers such as melanoma [14,15,16], ovarian [17] and colorectal cancers [18]. For example, miR-200c is up-regulated in melanoma lines compared to normal melanocytes [14] and in primary melanoma as compared to benign nevi [16]. Additionally, analysis of microRNA levels showed that miR200c was up-regulated in melanoma metastases to the lung, although down-regulated in those to the brain, as compared to primary lesions [15]. Taken together, this data suggests that miR200c is differentially regulated in melanoma and may play a role in disease initiation and/or progression. In order to investigate the functional effects of the miR-200 family in melanoma, we decided to test whether expression of miR-200 family members affects the ability of melanoma cells to engage in morphological switching and use different modes of migration to invade into a physiologic 3D collagen-I matrix. We confirmed that miR-200 members are up-regulated in melanoma, show they do not suppress invasion into 3D matrices and sometimes increase invasive capacity. Interestingly, while elevation of miR-200a levels led to the mesenchymal mode of cell migration, elevation of miR-200c levels led to the amoeboid mode of migration, highlighting new roles of this microRNA family in switching or plasticity of modes of tumor cell migration. microRNA-200 family members regulate modes of invasion in melanoma cells Individual members of the miR-200 family can be subdivided in two different ways according to geno (...truncated)